The objective of this proposal is to isolate and characterize the genes defective in Fanconi Anemia (FA). Patients with this hereditary disorder have a varied set of congenital malformations, bone marrow failure and an increased risk for the development of myeloid leukemia. Thus, the genes defective in FA will reveal information about developmental processes in addition to cancer biogenesis. We have shown that there are at least four complementation groups in FA (A to D) and have developed an episomal cDNA functional complementation method, that exploits FA hypersensitivity to DNA-crosslinking agents, to isolate the defective genes. Recently, we have successfully used this method to isolate the gene for FA(C) and found that it codes for a novel protein. It is thus important to establish its functional relationship to the genes in the other FA groups. Two main projects are proposed. First, we will use the same methodology to clone the genes defective in the A, B and D groups. Candidate cDNAs will be tested for their ability to specifically correct the cellular defect in the same FA cells. The assignment will be confirmed by finding mutations in FA patients of the same group. Second, we will characterize the FA genes and their protein products. We will map the cDNA in the human genome, isolate the gene and characterize the intron-exon structure. we will raise antibodies against the predicted protein and use these to establish its physical properties and cellular location. The cell, tissue and developmental expression of the gene will established by RNA and in situ hybridization analysis of human and rodent samples. This research will have major impact in our understanding of the basic defect in this complex disorder. In the first instance, it will be possible to classify the various patients according to complementation group and to establish correlations between the manifestation of their disease and the underlying genetic defects. Second, DNA based tests will be developed for accurate diagnosis as well as for carrier and prenatal testing. In the longer term, as knowledge about the genes and their expression is developed, it may be feasible to consider gene therapy as a method of treating FA patients.